Journal: Nar Molecular Medicine

Article Title: C G composition in transposon-derived genes is increased in FXD with perturbed immune system

doi: 10.1093/narmme/ugae015

Figure Lengend Snippet: Protein expressions of TE-derived genes responding viral infection were changed in FXD. (A, B) Protein expressions of TE-derived genes regulating immune responses (TLR3, IRF5, TSC1, CD46 and THOC1), viral responses (PLSCR1 and DXX17), oxidoreductase activity (P4HA2), and mitochondrial function and purine metabolism (NDUFS3) in the extracts from indicated LCLs were analyzed by western blots. eIF3β was used as the loading control. ( C ) Summary model is illustrated. Viral RNA with a high content of CG dinucleotides is integrated into the host genome by exploiting CG suppressor to decrease antigens and is retained as retrotransposons (TEs) (left panel). Some viral infections trigger hypoxia, which promotes conversion of O 2 to H 2 O 2 via XO (left panel). Inflammation induces XDH expression and conversion of XDH to XO, producing ROS including H 2 O 2 (left and right panels). In FXD in the present, increased substitution of A T to C G in TE-derived genes causes splicing changes, including in HLA genes (right panel). Increased CG dinucleotide changes expression of TE-derived genes regulating immune responses, suggesting that these genes had deceived CG suppression. Thus, maintaining high levels of C G content and CG dinucleotides in TE-derived genes likely stimulates XO activity and has maintained expression of TEs.

Article Snippet: The cells were lysed in a 5× volume of ice-cold Extraction Buffer I (10 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (pH 7.5), 1.5 mM magnesium chloride, 10 mM potassium chloride, 1 mM dithiothreitol and proteinase inhibitors) for 10 min, followed by incubation with 1% nonoxynol-40 for 5 min. Cytoplasmic extracts were obtained from supernatants after centrifugation at 2655 g for 5 min ( ) and were examined by western blotting with antibodies against MOCS2 (sc-377169, Santa Cruz), xanthine dehydrogenase/oxidase (35792, Cell Signaling Technology), eIF4E (sc-9976, Santa Cruz), TRL3 (83136–3-RR, proteintech), IRF-5 (sc-56714, Santa Cruz), TSC1 (6935, Cell Signaling Technology), P4HA2 (66604-1-Ig, proteintech), CD46 (sc-52647, Santa Cruz), NDUFS3 (sc-374282, Santa Cruz), THOC1 (sc-514123, Santa Cruz), PLSCR1 (11582-1-AP, proteintech), DDX17 (sc-398168, Santa Cruz) and eIF3β (sc-374156, Santa Cruz, RRID:AB_11008477 ).

Techniques: Derivative Assay, Infection, Activity Assay, Western Blot, Control, Expressing

Journal: Journal of Thoracic Disease

Article Title: Hepatocyte growth factor alleviates alcoholic cardiomyopathy through the Nrf2 pathway—a focus on iron metabolism

doi: 10.21037/jtd-2025-1111

Figure Lengend Snippet: Effects of exogenous HGF on ferroptosis in cardiomyocytes. (A) The effect of HGF on cell viability (CCK8); (B) the effect of HGF on GPX4; (C) the effect of HGF on LIP; (D) the effect of HGF on cell survival by calcein staining; (E) MDA concentrations of different HGF doses; (F) GPX4 concentrations of different HGF doses; (G) LIP levels of different HGF doses; (H) MDA concentrations following Nfe2l2 knockdown in ethanol-treated cells; (I) GPX4 concentrations following Nfe2l2 knockdown in ethanol-treated cells; (J) LIP levels following Nfe2l2 knockdown in ethanol-treated cells; (K) Cell survivals by calcein staining following Nfe2l2 knockdown in ethanol-treated cells. **, P<0.01; ns, P>0.05. CCK8, Cell Counting Kit-8; GPX4, glutathione peroxidase 4; HGF, hepatocyte growth factor; LIP, labile iron pool; MDA, malondialdehyde; MFI, mean fluorescence intensity; PBS, phosphate buffered saline; scramble, randomly interrupted sequence; siRNA, small interfering RNA.

Article Snippet: The Bicinchoninic Acid protein assay kit (71285-M), calcein-AM (206700) and 3-hydroxy-1,2-dimethyl-4(1H)-pyridone (379409) from Sigma-Aldrich (St. Louis, USA); the Trizol reagent, RIPA protein lysis buffer, malondialdehyde (MDA) kit (S0131S), Cell Counting Kit-8 (CCK8), calcein staining kit (C2013S) and hematoxylin-eosin (HE) staining kit (C0105S) from Beyotime (Shanghai, China); the glutathione peroxidase 4 (GPX4) (CSB-EL009869RA) and HGF (CSB-E07346r) enzyme-linked immunosorbent assay (ELISA) kit from Cusabio (Wuhan, China).

Techniques: Staining, Knockdown, Cell Counting, Fluorescence, Saline, Sequencing, Small Interfering RNA

Journal: International Journal of Molecular Medicine

Article Title: α-1,3-Fucosyltransferase-VII siRNA inhibits the expression of SLe x and hepatocarcinoma cell proliferation

doi: 10.3892/ijmm.2018.3850

Figure Lengend Snippet: Knocking down FUT7 affects cell survival via various intracellular signaling mediators. MHCC97 cells were transfected with FUT7 siRNAs. After 1 h, inhibitors of signaling molecules were added to the culture medium, and the cells were cultured for a further 48 h for RNA analysis and 72 h for protein analysis. (A) mRNA levels of FUT7 were quantified by reverse transcription-quantitative polymerase chain reaction following treatment with inhibitors. (B) Following treatment with the inhibitors, cell survival was quantified using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H tetrazolium bromide assay in the THLE-2 normal liver cells and MHCC97 hepatocarcinoma cells. (C) Cells were permeabilized and centrifuged to isolate the cytosolic proteins and particulate-conjugated proteins. Results of western blot analysis of the target proteins are shown with profiles. (D) Quantitative analysis of relative protein levels. * P<0.05 (n=3), vs. Control; ** P<0.01 (n=3), vs. Control. FUT, fucosyltransferase; Ctrl, control (no inhibitor); ErkI, extracellular signal-regulated kinase inhibitor; siRNA, small interfering RNA; siNC, scramble-siRNA transfected cells; PLCγ, phospholipase Cγ; PLCγ1-C, PLCγ1 in cytoplasm; PLCγ1-M, PLCγ1 conjugated with plasma membrane; p-PLCγ1, phosphorylated PLCγ1 conjugated with plasma membrane; Control, untransfected cells.

Article Snippet: The PVDF membranes were then incubated with 5% non-fat milk for 1 h at room temperature, and then incubated with anti-FUT7 (1:1,000, cat. no. MAB64091), anti-PLCγ1 (1:500, cat. no. MAB8137) and anti-phosphorylated PLCγ1 (1:500, cat. no. MAB74541) which were purchased from Bio-Techne China (Shanghai, China), anti-FUT6 (1:1,000, cat. no. NBP1-57936; Novus Biologicals, LLC, Littleton, CO, USA), or anti-β-actin antibody (1:1,000, cat. no. MAB8929; Bio-Techne China) at 4°C overnight.

Techniques: Transfection, Cell Culture, Real-time Polymerase Chain Reaction, Western Blot, Small Interfering RNA, Membrane

Journal: Cell Death & Disease

Article Title: Iron-overloaded follicular fluid increases the risk of endometriosis-related infertility by triggering granulosa cell ferroptosis and oocyte dysmaturity

doi: 10.1038/s41419-022-05037-8

Figure Lengend Snippet: A – C Levels of total iron, hepcidin, and transferrin in EMFF ( n = 15) and COFF ( n = 15). Data are expressed as means ± SD and analyzed by Student’s t test. D Results of mouse granulosa cells proliferation under different intervention conditions (each group in the figure is compared with COFF group). DFO, iron chelators; FER, ferroptosis inhibitor; NEC, necrosis inhibitor; ZDF, apoptosis inhibitor; ME, autophagy inhibitor. Data are expressed as means ± SD and analyzed by one-way ANOVA. E – H Comparison of ferritinophagy-related proteins FTH1, NCOA4, and ATG5 between human granulosa cells of infertile patients with EMs (EMGC) and of control group (COGC). The expression of β-actin was used as an internal control. Data are expressed as means ± SD and analyzed by Student’s t test. I – L Detection of ferroptosis-related indicators iron, GSH, GPX4, and MDA in COGC and EMGC. Data are expressed as means ± SD and analyzed by Student’s t test. M Representative images of the mitochondrial morphology of mouse granulosa cells intervened by COFF and EMFF were observed under TEM. Yellow arrows indicate mitochondrion. Scale bar = 1.0 µm. Scale bar = 5.0 µm. N Representative images of ROS and ferrous ion fluorescence staining after COFF and EMFF intervention in mouse granulosa cells. Scale bar = 100 µm. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, and ns, no significance.

Article Snippet: GPX4 assay kit (CSB-EL009869HU) was obtained from CUSABIO (Hubei, China).

Techniques: Comparison, Control, Expressing, Fluorescence, Staining

Journal: Cell Death & Disease

Article Title: Iron-overloaded follicular fluid increases the risk of endometriosis-related infertility by triggering granulosa cell ferroptosis and oocyte dysmaturity

doi: 10.1038/s41419-022-05037-8

Figure Lengend Snippet: A The average body weight of the pregnant mice was calculated from control (CON, n = 10), EMs model (EMs, n = 10), EMs combined with iron overload model (IRON, n = 10), DFO treatment after IRON model (IRON + DFO, n = 10), and VITE treatment after IRON model (IRON + VITE, n = 10). B Representative photographs of the uterus after pregnancy in each of the five groups of female mice. C , D Total litter size and specific number of litters per mouse in five groups ( n = 10). E For Prussian blue, H&E, and GPX4 immunohistochemical staining, ovarian tissues were observed from the five groups of mice. Scale bar = 50 µm; Scale bar = 200 µm; Scale bar = 20 µm. F Western blot analysis of TP53 expression in the ovarian tissues of mice in each group. Expression of GAPDH was used as an internal control.

Article Snippet: GPX4 assay kit (CSB-EL009869HU) was obtained from CUSABIO (Hubei, China).

Techniques: Control, Immunohistochemical staining, Staining, Western Blot, Expressing